Fluorescence images can be generated in-vivo for imaging of molecular functions and gene expression in live biological tissues. Fluorescence imaging of small animals has been used in biological research, including research in drug discovery and in investigation of fluorescent reporter technologies. Fluorescence imaging has also been used to study various human tissues, for example, tissues exhibiting epithelial diseases, the human breast, joints, and human teeth.
Conventionally, fluorescent light has been used for high-resolution imaging of histological slices of biological tissue using so-called fluorescence microscopy. Fluorescence microscopy is used to provide relatively high-resolution images. However, tissue sectioning used in conventional fluorescence microscopy is limited to slice thicknesses (i.e., tissue depths) on the order of half a millimeter, and therefore, conventional fluorescence microscopy is not appropriate for imaging through entire organs or through the whole human body.
In order to provide images a bit deeper into tissue, conventional systems and techniques have used light sources and fluorochromes that emit near infrared light. The near infrared light is selected because near infrared light has low absorption and can penetrate several centimeters into biological tissue. Near infrared light is used in a variety of optical imaging systems and techniques.
Fluorescent light can be emitted from a tissue in response to an excitation light source transmitting excitation light into the tissue. The excitation light excites the emission of fluorescent light from fluorochromes within the tissue.
Similarly, bioluminescence imaging has been used to image into tissues. The difference between fluorescence and bioluminescence imaging is that, for bioluminescence imaging, no excitation light source is required to cause emission of bioluminescent light. Emission of bioluminescent light in bioluminescence imaging is caused by a chemi-luminescent reaction within the tissue, resulting from transgenes.
The most common macroscopic technique that is conventionally used for fluorescence imaging is fluorescence reflectance imaging (FRI), which is also referred to herein as fluorescence epi-illumination imaging (FEI).
Epi-illumination light sources and epi-illumination imaging are further described below. In general, an epi-illumination light source generates light that is directed toward and then reflects from a surface of biological tissue and/or that propagates into the biological tissue and reflects from internal structures and/or surfaces of the biological tissue. To form an epi-illumination image, image light is collected generally on the same side of the tissue as the epi-illumination light source.
An FEI system transmits light onto and/or into biological tissue and collects the fluorescence light that is emitted back from the tissue, including light that is emitted back from within the tissue. In fluorescence epi-illumination imaging, excitation light (for example, near-infrared light) from an epi-illumination light source is used to illuminate the tissue. The epi-illumination light source is used to excite fluorochromes within the tissue that, in turn, emit fluorescent light. In some arrangements, the emitted light is visible light. In other arrangements, the emitted light is near infra red light. The emitted light can be visually inspected or it can be captured with a CCD camera or other photon detector positioned generally on the same side of the tissue as the epi-illumination light source. Bioluminescence imaging can be similar to fluorescent epi-illumination imaging, but bioluminescence is generated without an epi-illumination light source.
As described above, conventional fluorescence imaging with near-infrared light provides images having relatively low resolution and only small penetration (2-3 mm) of tissue. Higher resolution is achieved when spectral information is utilized and “umixed.”
A second method, which has not yet been utilized for research using small animals, but which has found applications in optical breast imaging, uses a transillumination light source to generate transillumination images. Similar to the above-described epi-illumination light source, a transillumination light source generates light that propagates into the tissue. However, unlike epi-illumination light, the transillumination light propagates entirely through the tissue. In transillumination imaging, image light is collected generally on the opposite side of the tissue from the transillumination light source.
Similar to that described above for fluorescence epi-illumination imaging, in fluorescence transillumination imaging, excitation light (for example, near infra red light) from a transillumination light source is used to illuminate a tissue. The excitation light propagates into the tissue, exciting the emission of fluorescent light from within the tissue. However, in contrast to the above-described fluorescence epi-illumination arrangement, in fluorescence transillumination imaging, a CCD camera or other photon detector is positioned generally on the opposite side of the tissue from the transillumination light source. In some arrangements, the emitted light is near infrared light. Fluorescence transillumination imaging (FTI) has been used to visualize functional characteristics of cardiac muscle and in dental diagnostic practice.
In some transillumination arrangements, the transillumination light source and the light detector lie on a virtual line passing through the tissue. In some arrangements the virtual line is generally perpendicular to the tissue and, in other arrangements, the virtual line is not generally perpendicular to the tissue.
Fluorescence epi-illumination imaging (FEI), fluorescence transillumination imaging (FTI), and bioluminescence imaging (BI) are forms of “planar” imaging, which provide two-dimensional images.
More advanced optical imaging systems and methods have been developed, which utilize tomographic methods. These systems and methods operate by obtaining photonic measurements at different projections (i.e., angles) to the tissue and combining the measurements using a tomographic algorithm. Tomography can provide a more accurate image than the above-described forms of planar imaging. Advantages of tomography include an ability for image quantification, an ability to provide two-dimensional or three-dimensional images, an ability to provide three-dimensional imaging with feature depth measurements, and higher sensitivity and higher resolution as compared to planar imaging. In some applications, tomography has been used in-vivo to measure enzyme regulation and treatment response to drugs. In these applications, tomography provides superior imaging performance to planar imaging. However, tomography is more complex than planar imaging, requiring more advanced instrumentation, requiring multiple illumination points (projections), which can require multiple light sources, and requiring advanced theoretical methods for modeling photon propagation in tissues.